80
81
Materials
General chemicals were of the best grade available, supplied by Tokyo Chemical Industries, Wako Pure Chemical, Aldrich Chemical Co., Dojindo, Watanabe Chemical Industries and Invitrogen, and were used without further purification. HeLa cells were purchased from American Type Culture Collection (ATCC). MEF cells stably expressing Vamp7-Venus were kindly gifted from Prof.
Noboru Mizushima’s lab (The University of Tokyo, Graduate School of Medicine). COS-1 cells were kindly gifted from Prof. Hiroyuki Arai’s lab (The University of Tokyo, Graduate School of Pharmaceutical Sciences)
Instruments
NMR spectra were recorded on a JEOL JNM-LA300 instrument at 300 MHz for 1H NMR and at 75 MHz for 13C NMR, or a JEOL JNM-LA400 instrument at 400 MHz for 1H NMR and at 100 MHz for 13C NMR. Mass spectra (MS) were measured with a JEOL JMS-T100LC AccuToF (ESI). HPLC analysis was performed on an Inertsil ODS-3 (4.6 × 250 mm) column (GL Sciences Inc.) using an HPLC system composed of a pump (PU-980, JASCO) and a detector (MD-2015, JASCO).
Preparative HPLC was performed on an Inertsil ODS-3 (10.0 × 250 mm) column (GL Sciences Inc.) using an HPLC system composed of a pump (PU-2080, JASCO) and a detector (MD-2015 or FP-2025, JASCO).
UV-Vis Absorption and fluorescence spectroscopy
UV-Visible spectra were obtained on a Shimadzu UV-1650. Fluorescence spectroscopic studies were performed on a Hitachi F4500. The slit width was 5 nm for both excitation and emission. The photomultiplier voltage was 700 V. Absolute Quantum Yields were measured by a Hamamatsu Photonics Quantaurus QY. Relative fluorescence quantum efficiencies were obtained by comparing the area under the emission spectrum of the test sample with standard samples. Values were calculated according to the following equation.
Φx/Φst = [Ast/Ax][nx2/nst2][Dx/Dst]
where st: standard; x: sample; A: absorbance at the excitation wavelength; n: refractive index; D:
area under the fluorescence spectra on an energy scale
82
Computation Details
All calculations were performed at the Density Functional Theory (DFT), by means of the B3LYP50-52 functional level as implemented in Gaussian 0953. The 6-31+G(d) basis set was used for all atoms. The number of imaginary frequencies is 0 for all structures.
Preparation of Cells
HeLa cells, MEF cells and COS-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Corp., Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), 1% penicillin streptomycin (Invitrogen). Cells were cultured at 37 °C in a 5/95 CO2/air incubator.
Lysosomal pH Imaging in MEF Cells with SiRpH5-Dex
1.6×104 MEF cells stably expressing Vamp7-Venus were seeded on 8 chamber plate (NUNCTM) and cultured for 1 day before imaging. Cells were incubated in 200 µL of DMEM (10% FBS) containing 200 µg/ml SiRpH5-Dex for 2 hr at 37oC, then washed three times with DMEM and further incubated in DMEM for 3 hr at 37oC and imaged. The fluorescence imaging were operated using a Leica Application Suite Advanced Fluorescence (LAS-AF) instrument with a TCS SP5 and 63× oil immersion objective lens. The light source was white-light laser. The excitation wavelength and the emission wavelength were 510 nm / 530-560 nm for Vamp7-Venus and 580 nm, 670 nm/ 690-750 nm for SiRpH5-Dex.
Endosomal pH Imaging in COS-1 Cells with SiRpH5-Tfn
5.0×104 COS-1 cells were seeded on 35 mm poly-L-lysine-coated glass-bottomed dishes (Matsunami Glass Ind., LTD.), transfected GFP-3xFYVE or EGFP-Rab11 with lipofectamine 2000 and cultured for 1 day before imaging. Cells were incubated in 1 mL of HBSS containing 25 µg/ml SiRpH5-Tfn for 20 min at 4oC, then washed twice with ice cold HBSS and imaged at 37oC. The fluorescence imaging were operated using a Leica Application Suite Advanced Fluorescence (LAS-AF) instrument with a TCS SP5 and 63× oil immersion objective lens. The light source was white-light laser. The excitation wavelength and the emission wavelength were 488 nm / 500-530 nm for GFP-3xFYVE, EGFP-Rab11 and 580 nm, 660 nm/ 680-750 nm for SiRpH5-Tfn.
83
Labeling of Dextran with SiRpH3-SE
Each solution of 10 kDa aminodextran (1.42 mg, 142 nmol) in 284 µL of 100 mM borate buffer (pH 8.0) and 1, 3, 9, 15 eq. of SiRpH3-SE in 57 µL of DMSO were mixed and stirred at room temperature for 1.5 hr. Reaction mixtures were purified by size exclusion PD-10 columns using water mobile phase and lyophilized to yield SiRpH3-Dextrans.
Labeling of Dextran with SiRpH5-PEG
6-SE
Each solution of 10 kDa aminodextran (1.30 mg, 130 nmol) in 460 µL of 100 mM NaHCO3 aq. (pH 8.4) and 3.8, 7.5, 11.3 eq. of SiRpH5-PEG6-SE in 160 µL of DMSO were mixed and stirred at room temperature for 2 hr. Reaction mixtures were purified by size exclusion PD-10 columns using PBS mobile phase, desalted with PD-10 column using water mobile phase and lyophilized to yield SiRpH5-Dextrans.
Labeling of Holo-transferrin with SiRpH5-PEG
6-SE
Each solution of 80 kDa human Holo-transferrin (9.04 mg, 113 nmol) in 1 mL of 100 mM borate buffer (pH 8.0) and 8, 11 eq. of SiRpH5-PEG6-SE in 1 mL of 100 mM borate buffer (pH 8.0) containing 20% DMSO were mixed and stirred at room temperature for 1 hr. Reaction mixtures were purified by size exclusion PD-10 columns using PBS mobile phase, desalted with PD-10 column using water mobile phase and lyophilized to yield SiRpH5-Tfns.
Synthetic procedures and characterizations
To a solution of 3-bromo-N,N-diallylaniline54 (10) (1.21 g, 4.80 mmol) in toluene (20 mL) were added DMF (456 L, 6.24 mmol) and phosphorus oxychloride (534 L, 5.76 mmol) under argon atmosphere. The reaction mixture was stirred for 21h at 80°C. An aqueous solution of 2N NaOH was added while the system was immersed in a water bath and cooled, the system was stirred for 5 minutes, and the mixture was extracted with dichloromethane and washed with brine. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, ethanol (10
84
mL) and sodium borohydride (182 mg, 4.80 mmol) were added, and the system was stirred for 1h at room temperature. After the solvent had been distilled away under reduced pressure, water was added to the residue and the mixture was extracted using dichloromethane. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, and the residue was purified using column chromatography (silica gel, AcOEt/n-hexane) to afford 15 (1.15 g, 4.08 mmol, 85%).
1H NMR (300 MHz, CDCl3): δ 3.89-3.91 (m, 4H), 4.62 (d, J = 6.0 Hz, 2H), 5.12-5.19 (m, 4H), 5.76-5.88 (m, 2H), 6.60 (dd, J = 2.3, 8.6 Hz, 1H), 6.86 (d, J = 2.3 Hz, 1H), 7.20 (d, J = 8.6 Hz, 1H);
13C NMR (75 MHz, CDCl3) δ 52.69, 65.04, 111.31, 115.86, 116.31, 124.43, 136.95, 130.45, 133.02, 149.35; LRMS (ESI+) 264 [M–OH]+
To a solution of 3-bromoaniline (27) (2 g, 11.6 mmol) in DMF (8 mL) was added KI (1.5 g, 9.3 mmol). The reaction mixture was stirred for 26 h at 130°C. The mixture was concentrated in vacuo and H2O was added to it, then the aqueous layer was extracted with n-hexane. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 10% AcOEt / n-hexane) and further purified by GPC to afford 17 (620 mg, 2.46 mmol, 21%).
1H NMR (300 MHz, CDCl3): δ 1.90-2.01 (m, 4H), 2.69 (t, J = 6.6 Hz, 2H), 2.77 (t, J = 6.6 Hz, 2H), 3.08-3.15 (m, 4H), 6.63 (d, J = 8.0 Hz, 1H), 6.75 (d, J = 8.0 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 21.80, 21.91, 27.58, 28.48, 49.65, 50.00, 119.42, 120.55, 120.65, 122.90, 127.73, 144.54; HRMS (ESI+) Calcd for [M+H]+, 254.0367 Found, 254.0331 (–3.6 mmu)
To a solution of 6-bromo-1-methylindoline16 (28) (212 mg, 1.00 mmol) in DMF (5 mL) was added
85
phosphorus oxychloride (139 L, 1.5 mmol) under argon atmosphere. The reaction mixture was stirred for 14 h at 80°C. An aqueous solution of 2N NaOH was added while the system was immersed in a water bath and cooled, the system was stirred for 15 minutes, and the mixture was extracted with dichloromethane and washed with brine. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, methanol (10 mL) and sodium borohydride (38 mg, 1.00 mmol) were added, and the system was stirred for 1 h at room temperature. After the solvent had been distilled away under reduced pressure, water was added to the residue and the mixture was extracted using dichloromethane. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, and the residue was purified using column chromatography (silica gel, AcOEt/n-hexane) to afford 26 (224 mg, 0.926 mmol, 85%).
1H NMR (300 MHz, CDCl3): δ 1.81 (t, J = 5.9 Hz, 1H), 2.74 (s, 3H), 2.90 (t, J = 8.0 Hz, 2H), 3.35 (t, J = 8.0 Hz, 2H),4.63 (d, J = 5.9 Hz, 2H), 6.60 (s, 1H), 7.10 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 28.06, 35.68, 56.05, 65.02, 110.58, 121.83, 125.28, 128.20, 130.00, 154.02; HRMS (ESI+) Calcd for [M+H]+, 244.0160, Found, 244.0126 (–3.4 mmu)
To a solution of 3-bromoaniline (27) (2 g, 11.6 mmol) in CH3CN (35 mL) were added K2CO3 (4 g, 29.0 mmol), benzylbromide (3.45 mL, 29.1 mmol) and tetrabutylammonium iodide (429 mg, 1.16 mmol). The reaction mixture was stirred for 63 h at 80°C. The mixture was concentrated in vacuo and H2O was added to it, then the aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH2Cl2/n-hexane) to afford 29 (3.54 g, 10.1 mmol, 21%).
1H NMR (300 MHz, CDCl3): 4.61 (s, 4H), 6.63 (dd, J = 8.3, 2.7 Hz, 1H), 6.81 (d, J = 8.3 Hz, 1H), 6.89 (s, 1H), 6.99 (t, J = 8.3 Hz, 1H), 7.20-7.36 (m, 10H); 13C NMR (75 MHz, CDCl3) δ 54.03, 111.10, 115.11, 119.61, 123.50, 126.58, 127.10, 128.74, 130.42, 137.78, 150.48
86
To a solution of 29 (3.51 g, 9.97 mmol) in toluene (20 mL) were added DMF (946 µL, 13.0 mmol) and phosphorus oxychloride (1109 L, 12.0 mmol) under argon atmosphere. The reaction mixture was stirred for 15 h at 80°C. An aqueous solution of 2N NaOH was added while the system was immersed in a water bath and cooled, the system was stirred for 30 minutes, and the mixture was extracted with dichloromethane and washed with brine. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, ethanol (20 mL) and sodium borohydride (379 mg, 9.97 mmol) were added, and the system was stirred for 3 h at room temperature. After the solvent had been distilled away under reduced pressure, water was added to the residue and the mixture was extracted using dichloromethane. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, and the residue was purified using column chromatography (silica gel, AcOEt/n-hexane) to afford 20 (1.45 g, 3.79 mmol, 38%).
1H NMR (300 MHz, CDCl3): 4.58 (s, 2H), 4.60 (s, 4H), 6.63 (dd, J = 8.7, 2.5 Hz, 1H), 6.94 (d, J = 2.5 Hz, 1H), 7.15 (d, J = 8.7 Hz, 1H), 7.18-7.34 (m, 10H); 13C NMR (75 MHz, CDCl3) δ 54.02, 64.87, 111.54, 115.96, 124.50, 126.51, 127.01, 127.53, 128.72, 130.52, 137.61, 149.84; HRMS (ESI+) Calcd for [M+Na]+, 404.0626, Found, 404.0655 (+2.9 mmu)
To a solution of 3-bromoaniline (27) (2 g, 11.6 mmol) in DMF (20 mL) were added K2CO3 (7.07 g, 51.2 mmol), 1,4-dibromobutane (2.76 g, 12.8 mmol) and KI (10 mg, 60.2 µmol). The reaction mixture was stirred for 19 h at 100°C. The mixture was concentrated in vacuo and H2O was added to it, then the aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH2Cl2/n-hexane) to afford 30 (1.17 g, 5.18 mmol, 45%).
87
1H NMR (300 MHz, CDCl3): δ 1.97-2.01 (m, 4H), 3.22-3.27 (m, 4H), 6.45 (dd, J = 8.0, 2.2 Hz, 1H), 6.67 (s, 1H), 6.74 (d, J = 8.0 Hz, 1H), 7.04 (t, J = 8.0 Hz, 1H) ; 13C NMR (75 MHz, CDCl3) δ 25.41, 47.55, 110.23, 114.27, 117.98, 123.32, 130.25, 148.99; HRMS (ESI+) Calcd for [M+H]+, 226.0231, Found, 226.0202 (–2.9 mmu)
To a solution of 30 (511 mg, 2.26 mmol) in toluene (6 mL) were added DMF (214 µL, 2.94 mmol) and phosphorus oxychloride (251 L, 2.71 mmol) under argon atmosphere. The reaction mixture was stirred for 14 h at 80°C. An aqueous solution of 2N NaOH was added while the system was immersed in a water bath and cooled, the system was stirred for 30 minutes, and the mixture was extracted with dichloromethane and washed with brine. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, methanol (15 mL), CH2Cl2 (5 mL) and sodium borohydride (379 mg, 9.97 mmol) were added, and the system was stirred for 6 h at room temperature. After the solvent had been distilled away under reduced pressure, water was added to the residue and the mixture was extracted using dichloromethane. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, and the residue was purified using column chromatography (silica gel, AcOEt/n-hexane) to afford 23 (421 mg, 1.64 mmol, 73%).
1H NMR (300 MHz, CDCl3): δ 1.79 (t, J = 6.2 Hz, 1H), 1.98-2.03 (m, 4H), 3.24-3.28 (m, 4H), 4.64 (d, J = 6.2 Hz, 1H), 6.47 (dd, J = 8.1, 2.1 Hz, 1H), 6.74 (d, J = 2.1 Hz, 1H), 7.22 (d, J = 8.1 Hz, 1H) ; 13C NMR (75 MHz, CDCl3) δ 25.41, 47.60, 65.21, 110.76, 115.21, 124.49, 125.93, 130.62, 148.57; HRMS (ESI+) Calcd for [M+H]+, 256.0337, Found, 226.0365 (+2.8 mmu)
To a solution of 3-bromo-N,N-dimethylaniline (31) (516 mg, 2.58 mmol) in toluene (4 mL) were
88
added DMF (245 µL, 3.35 mmol) and phosphorus oxychloride (287 L, 3.10 mmol) under argon atmosphere. The reaction mixture was stirred for 20 h at 80°C. An aqueous solution of 2N NaOH was added while the system was immersed in a water bath and cooled, the system was stirred for 4 h, and the mixture was extracted with dichloromethane and washed with brine. The residue was purified using column chromatography (silica gel, AcOEt/n-hexane) to obtain 32 (470 mg, 2.06 mmol, 80%).
1H NMR (300 MHz, CDCl3): δ 3.08 (s, 6H), 6.64 (dd, J = 9.0, 2.7 Hz, 1H), 6.80 (d, J = 2.7 Hz, 1H), 7.80 (d, J = 9.0 Hz), 10.09 (s, 1H); 13C NMR (75 MHz, CDCl3) δ 40.04, 110.52, 114.80, 121.99, 129.66, 131.00, 154.48, 190.12
To a solution of 32 (433 mg, 1.90 mmol) in methanol (6 mL) was added sodium borohydride (74 mg, 1.90 mmol). The reaction mixture was stirred for 2 h at room temperature. The mixture was concentrated in vacuo and H2O was added to it, then the aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo.
The residue was purified by column chromatography (silica gel, AcOEt/n-hexane) to afford 21 (412 mg, 1.79 mmol, 87%).
1H NMR (300 MHz, CD2Cl2): δ 2.93 (s, 6H), 4.60 (d, J = 6.6 Hz, 1H), 6.65 (dd, J = 8.1, 3.0 Hz, 1H), 6.89 (d, J = 3.0 Hz, 1H), 7.24 (d, J = 8.1 Hz, 1H) ; 13C NMR (75 MHz, CDCl3) δ 40.37, 65.02, 111.40, 115.98, 124.36, 127.02, 130.35, 151.12; HRMS (ESI+) Calcd for [M+H]+, 230.0181, Found, 230.0162 (–1.9 mmu)
To a solution of 1-bromo-3-iodobenzene (33) (1.0 g, 3.53 mmol) in toluene (15 mL) were added
89
N-methylpiperazine (393 µL, 3.53 mmol), Pd(OAC)2 (79 mg, 0.353 mmol), BINAP (220 mg, 0.353 mmol) and Cs2CO3 (5.77 g, 17.7 mmol) under argon atmosphere. The reaction mixture was stirred for 18 h at 85°C. The reaction mixture was diluted with NaHCO3 aq., and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (NH silica gel, AcOEt/n-hexane) to afford 34 (540 mg, 2.12 mmol, 60%).
1H NMR (300 MHz, CDCl3): δ 2.35 (s, 3H), 2.56 (t, J = 5.1 Hz, 4H), 3.21 (t, J = 5.1 Hz, 4H), 6.83 (dd, J = 8.1, 2.1 Hz, 1H), 6.95 (d, J = 8.1 Hz, 1H), 7.03 (d, J = 2.1 Hz, 1H), 7.10 (t, J = 8.1 Hz, 1H);
13C NMR (75 MHz, CDCl3) δ 46.09, 48.57, 54.88, 114.27, 118.59, 122.10, 123.20, 130.26, 152.42;
HRMS (ESI+) Calcd for [M+H]+, 255.0497, Found, 255.04464 (–3.3 mmu)
To a solution of 34 (337 mg, 1.32 mmol) in DMF (5 mL) was added phosphorus oxychloride (2.45 mL, 26.4 mmol) under argon atmosphere. The reaction mixture was stirred for 18 h at 80°C. An aqueous solution of 2N NaOH was added while the system was immersed in a water bath and cooled, the system was stirred for 2h, and the mixture was extracted with dichloromethane and washed with brine. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, methanol (15 mL) and sodium borohydride (50 mg, 1.32 mmol) were added, and the system was stirred for 2 h at room temperature. After the solvent had been distilled away under reduced pressure, water was added to the residue and the mixture was extracted using dichloromethane. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, and the residue was purified by column chromatography (NH silica gel, AcOEt / n-hexane) and further purified by GPC to afford 24 (196 mg, 0.687 mmol, 52%).
1H NMR (300 MHz, CDCl3): δ 2.34 (s, 3H), 2.55 (t, J = 5.1 Hz, 4H), 3.18 (t, J = 5.1 Hz, 4H), 4.65 (s, 2H), 6.83 (dd, J = 8.1, 2.4 Hz, 1H), 7.08 (d, J = 2.4 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 45.87, 48.30, 54.65, 64.00, 114.79, 119.36, 123.42, 129.61, 130.81, 151.43; HRMS (ESI+) Calcd for [M+H]+, 285.0602, Found, 285.0613 (+1.1 mmu)
90
To a solution of 2-bromo-4-fluorobenzaldehyde (35) (2.41 g, 12.9 mmol) in DMF (30 mL) were added N-Bocpiperazine (2.19 g, 10.8 mmol) and K2CO3 (2.24 g, 16.2 mmol). The reaction mixture was stirred for 18 h at 100°C, diluted with NaHCO3 aq., and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was added methanol (30 mL) and sodium borohydride (494 mg, 13 mmol). The reaction mixture was stirred for 3 h at room temperature. After the solvent had been distilled away under reduced pressure, water was added to the residue and the mixture was extracted using dichloromethane. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, and the residue was purified by column chromatography (NH silica gel, AcOEt / n-hexane) to afford 25 (3.46 g, 9.33 mmol, 86%).
1H NMR (300 MHz, CDCl3): δ 1.48 (s, 9H), 2.02 (t, J = 6.6 Hz, 1H), 3.13 (t, J = 5.1 Hz, 4H), 3.56 (t, J = 5.1 Hz, 4H), 4.66 (d, J = 6.6 Hz, 2H), 6.85 (dd, J = 8.1, 2.2 Hz, 1H), 7.08 (d, J = 2.1 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 28.40, 48.89, 64.77, 80.05, 115.35, 120.09, 123.85, 130.03, 130.87, 151.76, 154.64; HRMS (ESI+) Calcd for [M+H]+, 371.0970, Found, 371.0922 (–4.8 mmu)
To a solution of 15 (73 mg, 0.260 mmol) and 11 (55 mg, 0.260 mmol) in CH2Cl2 (5 mL) was added BF3·OEt2 (65 µL, 0.520 mmol). The reaction mixture was stirred for 24 h at room temperature, diluted with water and extracted with CH2Cl2. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, and the residue was purified by column chromatography (silica gel, CH2Cl2/n-hexane) to afford 13 (116 mg, 0.244 mmol, 94%).
91
1H NMR (300 MHz, CDCl3): δ 2.73 (s, 3H), 2.83 (t, J = 8.1 Hz, 2H), 3.28 (t, J = 8.1 Hz, 2H), 3.87-3.88 (m, 4H), 3.97 (s, 2H), 5.14-5.20 (m, 4H), 5.76-5.89 (m, 2H), 6.64 (dd, J = 2.5, 8.2 Hz, 1H), 6.65 (s, 1H), 6.73 (s, 1H), 6.80 (d, J = 8.2 Hz, 1H), 6.90 (d, J = 2.5 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 28.36, 36.03, 40.14, 52.72, 56.28, 110.85, 111.64, 115.92, 116.27, 123.06, 125.49, 126.11, 127.05, 128.05, 130.17, 130.68, 133.52, 148.13, 153.03; LRMS (ESI+) 477 [M+H]+
To a solution of 15 (18.1 mg, 64.4 µmol) and 11 (14.5 mg, 72.5 µmol) in CH2Cl2 (5 mL) was added BF3·OEt2 (16 µL, 129 µmol). The reaction mixture was stirred for 8 h at 37oC, diluted with water and extracted with CH2Cl2. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, and the residue was purified by column chromatography (silica gel, AcOEt/n-hexane) to afford 37 (21.5 mg, 46.3 µmol, 72%).
1H NMR (300 MHz, CDCl3): δ 2.91 (s, 6H), 3.86-3.88 (m, 4H), 3.98 (s, 2H), 5.14-5.19 (m, 4H), 5.76-5.89 (m, 2H), 6.54 (dd, J = 2.5, 8.8 Hz, 1H), 6.59 (dd, J = 2.4, 8.5 Hz, 1H), 6.79 (d, J = 8.8 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.90 (d, J = 2.5 Hz, 1H), 6.93 (d, J = 2.4 Hz, 1H): 13C NMR (75 MHz, CDCl3): δ 39.8, 40.5, 52.7, 111.7, 111.8, 116.0, 116.2, 125.5, 125.6, 126.9, 127.1, 130.7, 130.8, 133.5, 148.1, 150.0; LRMS (ESI+) 465 [M+H]+
To a solution of 15 (25 mg, 89.0 µmol) and 11 (22 mg, 89.0 µmol) in CH2Cl2 (8 mL) was added BF3·OEt2 (22 µL, 178 µmol). The reaction mixture was stirred for 3 h at room temperature, diluted with water and extracted with CH2Cl2. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, and the residue was purified by column chromatography (silica gel, CH2Cl2/n-hexane) to afford 38 (40 mg, 77.5 µmol, 87%).
92
1H NMR (300 MHz, CDCl3): δ 1.89-2.03 (m, 4H), 2.64 (t, J = 6.6 Hz, 2H), 2.82 (t, J = 6.6 Hz, 2H), 3.05-3.12 (m, 4H), 3.86-3.88 (m, 4H), 3.95 (s, 2H), 5.14-5.19 (m, 4H), 5.76-5.89 (m, 2H), 6.49 (s, 1H), 6.54 (dd, J = 9.0, 3.0 Hz, 1H), 6.79 (d, J = 9.0 Hz, 1H), 6.90 (d, J = 3.0 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 21.98, 22.31, 27.52, 29.43, 40.88, 49.57, 50.11, 52.79, 111.73, 116.01, 116.31, 120.79, 121.27, 125.56, 125.63, 126.95, 127.25, 128.64, 128.70, 130.66, 133.68, 143.02, 148.10;
HRMS (ESI+) Calcd for [M+H]+, 515.0698 Found, 515.0712 (+1.5 mmu)
Compound 39 was synthesized as reported previously31.
Compound 40 was synthesized as reported previously31.
To a solution of 20 (40 mg, 105 µmol) and 11 (22 mg, 105 µmol) in CH2Cl2 (8 mL) was added BF3· OEt2 (27 µL, 210 µmol). The reaction mixture was stirred for 17 h at room temperature, diluted with water and extracted with CH2Cl2. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, and the residue was purified by GPC to afford 41 (52 mg, 90.2 µmol, 86%).
93
1H NMR (300 MHz, CDCl3): δ 2.72 (s, 3H), 2.83 (t, J = 8.1 Hz, 2H), 3.28 (t, J = 8.1 Hz, 2H), 3.96 (s, 2H), 4.59 (s, 4H), 6.56 (dd, J = 8.7, 2.7 Hz, 1H), 6.63 (s, 1H), 6.74-6.77 (m, 2H), 6.98 (d, J = 2.7 Hz, 1H), 7.20-7.35 (m, 10H); 13C NMR (75 MHz, CDCl3) δ 28.33, 35.98, 40.14, 54.09, 56.23, 110.84, 111.86, 116.01, 123.09, 125.55, 126.20, 126.65, 127.01, 127.66, 128.67, 130.16, 130.74, 138.05, 148.67, 153.05; HRMS (ESI+) Calcd for [M+H]+, 577.0677, Found, 577.0678 (+0.1 mmu)
To a solution of 21 (25 mg, 109 µmol) and 11 (23 mg, 109 µmol) in CH2Cl2 (6 mL) was added BF3· OEt2 (28 µL, 218 µmol). The reaction mixture was stirred for 17.5 h at room temperature, diluted with water and extracted with CH2Cl2. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, and the residue was purified by column chromatography (silica gel, AcOEt/n-hexane) to afford 42 (32 mg, 75.5 µmol, 69%).
1H NMR (300 MHz, CDCl3): δ 2.73 (s, 3H), 2.81 (t, J = 8.1 Hz, 2H), 2.91 (s, 6H), 3.28 (t, J = 8.1 Hz, 2H), 3.98 (s, 2H), 6.59 (dd, J = 8.8, 2.2 Hz, 1H), 6.65 (s, 1H), 6.70 (s, 1H), 6.86 (d, J = 2.2 Hz, 1H), 6.93 (d, J = 8.8 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 28.34, 36.02, 40.19, 40.50, 56.26, 110.86, 111.84, 116.20, 123.03, 125.56, 125.98, 127.22, 128.06, 130.13, 130.74, 149.98, 153.00; HRMS (ESI+) Calcd for [M+H]+, 425.0051, Found, 425.0022 (–2.9 mmu)
Compound 43 was synthesized as reported previously31.
94
To a solution of 23 (39 mg, 152 µmol) and 11 (32 mg, 152 µmol) in CH2Cl2 (10 mL) was added BF3·OEt2 (38 µL, 304 µmol). The reaction mixture was stirred for 13 h at room temperature, diluted with water and extracted with CH2Cl2. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, and the residue was purified by column chromatography (silica gel, CH2Cl2/n-hexane) to afford 44 (57.1 mg, 127 µmol, 83%).
1H NMR (300 MHz, CDCl3): δ 1.96-2.01 (m, 4H), 2.72 (s, 3H), 2.81 (t, J = 8.0 Hz, 2H), 3.22-3.30 (m, 6H), 3.98 (s, 2H), 6.42 (dd, J = 8.1, 2.1 Hz, 1H), 6.65 (s, 1H), 6.69 (s, 1H), 6.78 (d, J = 2.1 Hz, 1H), 6.85 (d, J = 8.1 Hz, 1H) ; 13C NMR (75 MHz, CDCl3) δ 25.41, 28.34, 36.03, 40.19, 47.58, 56.26, 110.79, 110.98, 115.14, 122.97, 125.57, 125.85, 125.95, 128.29, 130.07, 130.88, 147.33, 152.92; HRMS (ESI+) Calcd for [M+H]+, 451.0208, Found, 451.0222 (+1.4 mmu)
To a solution of 25 (41 mg, 110 µmol) and 11 (23 mg, 108 µmol) in CH2Cl2 (5 mL) was added BF3· OEt2 (28 µL, 220 µmol). The reaction mixture was stirred for 6 h at room temperature, diluted with water and extracted with CH2Cl2. The organic layer was dried using Na2SO4, the solvent was distilled away under reduced pressure, and the residue was purified by column chromatography (silica gel, AcOEt/n-hexane) to afford 45 (53 mg, 93.7 µmol, 87%).
1H NMR (300 MHz, CDCl3): δ 1.48 (s, 9H), 2.73 (s, 3H), 2.82 (t, J = 8.1 Hz, 2H), 3.10 (t, J = 5.1 Hz, 4H), 3.30 (t, J = 8.1 Hz, 2H), 3.56 (t, J = 5.1 Hz, 4H), 4.00 (s, 2H), 6.65 (s, 1H), 6.70 (s, 1H), 6.77 (dd, J = 8.7, 2.1 Hz, 1H), 6.88 (d, J = 8.7 Hz, 1H), 7.13 (d, J = 2.1 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ 28.31, 28.40, 35.96, 40.38, 49.13, 56.22, 79.95, 110.81, 115.64, 120.25, 123.13, 125.36, 126.09, 127.47, 130.20, 130.77, 131.20, 150.52, 153.13, 154.65; HRMS (ESI+) Calcd for [M+H]+, 566.0841, Found, 566.0850 (+0.9 mmu)
95
Compound 59 was synthesized from 37 as reported previously55.
To a solution of 13 (207 mg, 0.434 mmol) in anhydrous THF (10 mL) was added 1 M THF solution of sec-BuLi (0.91 mL, 0.91 mmol) at –78 °C under argon atmosphere. The reaction mixture was stirred for 20 min at the same temperature, then dichlorodimethylsilane (112 mg, 0.868 mmol) in anhydrous THF (5 mL) was added to it and the mixture was warmed to room temperature and stirred for 1 h. 2 N HCl aq. was added to the mixture to quench the reaction, and the mixture was neutralized with NaHCO3 aq. The whole was extracted with CH2Cl2. The organic layer was collected, washed with brine, dried over Na2SO4 and evaporated to dryness. The residue was dissolved in acetone (30 mL) at 0 °C and KMnO4 (275 mg, 1.74 mmol) was added in small portions over 3 h with stirring at the same temperature. Then the mixture was filtered through celite filter and the solution was evaporated to dryness. The residue was purified by column chromatography (silica gel, AcOEt/n-hexane) to afford 50 (30 mg, 77.3 µmol, 18%).
1H NMR (300 MHz, CDCl3): δ 0.43 (s, 6H), 2.90 (s, 3H), 3.05 (t, J = 8.5 Hz, 2H), 3.47 (t, J = 8.5 Hz, 2H), 4.02-4.03 (m, 4H), 5.18-5.23 (m, 4H), 5.82-5.94 (m, 2H), 6.50 (s, 1H), 6.80-6.86 (m, 2H), 8.21 (s, 1H), 8.34 (d, J = 9.0 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ -1.17, 28.03, 34.55, 52.65, 54.79, 107.89, 107.93, 113.37, 114.65, 116.52, 120.40, 126.03, 129.98, 131.47, 131.59, 132.12, 133.04, 140.12, 140.27, 150.00, 154.80, 185.05; HRMS (ESI+) Calcd for [M+H]+, 389.2049 Found, 389.2069 (+2.0 mmu)
96
To a solution of 50 (506 mg, 1.30 mmol) in CH2Cl2 (20 ml) were added 1,3-dimethylbarbituric acid (406 mg, 2.60 mmol) and Pd(PPh3)4 (225 mg, 0.145 mmol) under argon atmosphere. The reaction mixture was stirred for 19 h at 35°C. The reaction was quenched with sat. NaHCO3 aq., and the aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with sat.
NaHCO3 aq. and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, AcOEt/CH2Cl2) to afford 51 (354 mg, 1.15 mmol, 88%).
1H NMR (300 MHz, CDCl3): δ 0.43 (s, 6H), 2.90 (s, 3H), 3.04 (t, J = 8.2 Hz, 2H), 3.47 (t, J = 8.2 Hz, 2H), 6.49 (s, 1H), 6.76-6.82 (m, 2H), 8.20 (s, 1H), 8.31 (d, J = 8.1 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ -1.28, 27.96, 34.44, 54.73, 107.79, 107.84, 116.12, 117.51, 126.10, 131.15, 131.84, 132.20, 140.12, 140.80, 148.83, 154.91, 185.13; HRMS (ESI+) Calcd for [M+H]+, 309.1423 Found, 389.1426 (+0.3 mmu)
To a solution of 51 (8.7 mg, 28.2 µmol) in anhydrous THF (5 mL) was added 1 M THF solution of o-tolylmagnesium bromide (2.8 mL, 2.82 mmol) and the mixture was refluxed at 80°C for 2 h under argon atmosphere. After it had cooled to room temperature, 2 N HCl aq. was added to it. The mixture was diluted with Sat. NaHCO3 aq., and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by HPLC (eluent, 48% CH3CN/0.1% TFA aq. (0 min) to 56% CH3CN/0.1% TFA aq. (20 min); flow rate
= 5.0 mL/min) to afford 60 (5.3 mg, 10.7 µmol, 38%).
1H NMR (300 MHz, CD2Cl2): δ 0.54 (s, 3H), 0.56 (s, 3H). 2.03 (s, 3H), 2.96 (t, J = 8.0 Hz, 2H), 3.21
97
(s, 3H), 3.83 (t, J = 8.0 Hz, 2H), 6.58 (d, J = 8.7 Hz, 1H), 6.79 (s, 1H), 6.91 (s, 1H), 6.95 (d, J = 8.7 Hz, 1H), 7.09 (d, J = 7.2 Hz, 1H), 7.29-7.43 (m, 4H) ); 13C NMR (100 MHz, CD3OD) δ -1.62, -1.34, 19.43, 26.80, 33.93, 56.04, 116.25, 117.14, 123.40, 126.85, 128.62, 129.81, 129.87, 130.08, 131.34, 134.54, 135.44, 136.88, 140.66, 141.80, 147.77, 154.49, 156.57, 159.59, 168.63; HRMS (ESI+) Calcd for [M+H]+, 383.1944 Found, 383.1484 (+4.0 mmu)
Compound 61 was synthesized from 40 as reported previously31.
To a solution of 5431 (20 mg, 46.4 µmol) in anhydrous THF (5 mL) was added 1 M THF solution of o-tolylmagnesium bromide (10 mL, 10 mmol) and the mixture was refluxed at 65°C for 13 h under argon atmosphere. After it had cooled to room temperature, 2 N HCl aq. was added to it. The mixture was diluted with Sat. NaHCO3 aq., and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by HPLC (eluent, 48% CH3CN/0.1% TFA aq. (0 min) to 80% CH3CN/0.1% TFA aq. (20 min); flow rate
= 25.0 mL/min) to afford 62 (16 mg, 27.6 µmol, 60%).
1H NMR (400 MHz, CDCl3): δ 0.54 (s, 3H), 0.55 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H), 1.43 (s, 3H), 1.45 (s, 3H), 1.51 (s, 3H), 2.05 (s, 3H), 3.14 (s, 3H), 3.47 (q, J = 7.2 Hz, 2H), 5.29 (s, 1H), 6.63 (s, 1H), 6.85 (s, 1H), 7.06-7.08 (m, 2H), 7.27-7.42 (m, 5H); 13C NMR (100 MHz, CDCl3) δ –1.38, –1.14, 13.86, 17.61, 19.41, 29.28, 32.10, 38.40, 59.43, 112.53, 115.47, 117.65, 118.38, 122.44, 125.56, 126.26, 127.54, 128.69, 128.92, 129.80, 130.07, 131.39, 135.67, 138.84, 148.24, 149.09, 155.96, 161.05, 161.39, 167.36; HRMS (ESI+) Calcd for [M]+, 465.2726, Found, 465.2676 (–4.1 mmu)
98
To a solution of 42 (354 mg, 0.835 mmol) in anhydrous THF (20 mL) was added 1 M THF solution of sec-BuLi (1.7 mL, 1.7 mmol) at –78 °C under argon atmosphere. The reaction mixture was stirred for 30 min at the same temperature, then dichlorodimethylsilane (118 µL, 1.02 mmol) in anhydrous THF (3 mL) was added to it and the mixture was warmed to room temperature and stirred for 5 h. 2 N HCl aq. was added to the mixture to quench the reaction, and the mixture was neutralized with NaHCO3 aq. The whole was extracted with CH2Cl2. The organic layer was collected, washed with brine, dried over Na2SO4 and evaporated to dryness. The residue was dissolved in acetone (30 mL) at 0 °C and KMnO4 (196 mg, 1.25 mmol) was added in small portions over 3 h with stirring at the same temperature. Then the mixture was filtered through celite filter and the solution was evaporated to dryness. The residue was purified by column chromatography (silica gel, AcOEt/n-hexane) and GPC. The residue was dissolved in anhydrous THF (10 mL), added 1 M THF solution of o-tolylmagnesium bromide (10 mL, 10 mmol) and the mixture was refluxed at 80°C for 5 h under argon atmosphere. After it had cooled to room temperature, 2 N HCl aq. was added to it. The mixture was diluted with Sat. NaHCO3 aq., and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by HPLC (eluent, 52% CH3CN/0.1% TFA aq. (0 min) to 72% CH3CN/0.1% TFA aq. (20 min); flow rate
= 25.0 mL/min) to afford 63 (25 mg, 47.7 µmol, 6%).
1H NMR (400 MHz, CD2Cl2): δ 0.47 (s, 3H), 0.50 (s, 3H), 1.95 (s, 3H), 2.88 (t, J = 7.2 Hz, 2H), 3.14 (s, 6H), 3.18 (s, 3H), 3.80 (t, J = 7.2 Hz, 2H), 6.46 (dd, J = 9.2, 2.0 Hz, 1H), 6.71 (s, 1H), 6.90-6.91 (m, 2H), 7.00-7.01 (m, 2H), 7.24-7.37 (m, 3H); 13C NMR (100 MHz, CD2Cl2) δ –1.03, –0.72, 19.69, 26.75, 34.37, 40.88, 55.73, 113.92, 115.94, 120.30, 126.28, 128.12, 129.34, 129.52, 129.78, 130.90, 134.33, 134.51, 136.41, 140.82, 146.77, 153.76, 154.28, 158.74, 168.76; HRMS (ESI+) Calcd for [M]+, 411.2257, Found, 411.2219 (–3.8 mmu)
99
To a solution of 5331 (10 mg, 27.4 µmol) in anhydrous THF (5 mL) was added 1 M THF solution of o-tolylmagnesium bromide (10 mL, 10 mmol) and the mixture was refluxed for 5 h under argon atmosphere. After it had cooled to room temperature, 2 N HCl aq. was added to it. The mixture was diluted with Sat. NaHCO3 aq., and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified column chromatography (silica gel, MeOH/CH2Cl2) and HPLC (eluent, 56% CH3CN/0.1% TFA aq. (0 min) to 72% CH3CN/0.1% TFA aq. (20 min); flow rate = 5.0 mL/min) to afford 64 (8 mg, 14.5µmol, 53%).
1H NMR (300 MHz, CD3CN): δ 0.55 (s, 3H), 0.56 (s, 3H), 1.21 (t, J = 7.1 Hz, 6H), 2.02 (s, 3H), 2.90 (t, J = 8.1 Hz, 2H), 3.23 (s, 3H), 3.58 (q, J = 7.1 Hz, 4H), 3.85 (t, J = 8.1 Hz, 2H), 6.63 (dd, J = 9.9, 2.6 Hz, 1H), 6.72 (s, 1H), 6.88 (d, J = 9.9 Hz, 1H), 7.09 (t, J = 7.2 Hz, 1H), 7.15 (s, 1H), 7.20 (d, J = 2.6 Hz, 1H), 7.34-7.48 (m, 3H); 13C NMR (100 MHz, CD3OD) δ –1.52, –1.24, 13.00, 19.41, 26.88, 33.88, 46.37, 55.96, 114.52, 116.79, 121.12, 126.85, 128.18, 129.87, 129.98, 130.13, 131.33, 134.29, 135.31, 136.95, 140.63, 141.28, 147.48, 152.86, 154.14, 159.43, 168.22; HRMS (ESI+) Calcd for [M]+, 439.2570, Found, 439.2522 (–4.8 mmu)
To a solution of 44 (336 mg, 0.747 mmol) in anhydrous THF (25 mL) was added 1 M THF solution of sec-BuLi (1.64 mL, 1.64 mmol) at –78 °C under argon atmosphere. The reaction mixture was stirred for 20 min at the same temperature, then dichlorodimethylsilane (134 µL, 1.12 mmol) in anhydrous THF (5 mL) was added to it and the mixture was warmed to room temperature and stirred
100
for 2 h. 2 N HCl aq. was added to the mixture to quench the reaction, and the mixture was neutralized with NaHCO3 aq. The whole was extracted with CH2Cl2. The organic layer was collected, washed with brine, dried over Na2SO4 and evaporated to dryness. The residue was dissolved in acetone (30 mL) at 0 °C and KMnO4 (472 mg, 2.99 mmol) was added in small portions over 2 h with stirring at the same temperature. Then the mixture was filtered through celite filter and the solution was evaporated to dryness. The residue was purified by column chromatography (silica gel, CH2Cl2/n-hexane) to afford 55 (21 mg, 58.0 µmol, 8%).
1H NMR (300 MHz, CDCl3): δ 0.45 (s, 6H), 2.03-2.07 (m, 4H), 2.90 (s, 3H), 3.05 (t, J = 8.0 Hz, 2H), 3.39-3.50 (m, 6H), 6.51 (s, 1H), 6.64 (d, J = 2.1 Hz, 1H), 6.69 (dd, J = 8.1, 2.1 Hz, 1H), 8.22 (s, 1H), 8.39 (d, J = 8.1 Hz, 1H) ; 13C NMR (100 MHz, CDCl3) δ 25.46, 28.06, 34.59, 47.45, 54.84, 107.95, 113.08, 114.08, 126.00, 129.10, 131.62, 131.80, 132.12, 140.07, 140.54, 148.95, 154.76, 185.16;
HRMS (ESI+) Calcd for [M+H]+, 363.1855, Found, 363.1893 (–3.8 mmu)
To a solution of 55 (15 mg, 41.4 µmol) in anhydrous THF (20 mL) was added 1 M THF solution of o-tolylmagnesium bromide (1 mL, 1 mmol) and the mixture was refluxed for 4 h under argon atmosphere. After it had cooled to room temperature, 2 N HCl aq. was added to it. The mixture was diluted with Sat. NaHCO3 aq., and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by HPLC (eluent, 64% CH3CN/0.1% TFA aq. (0 min) to 80% CH3CN/0.1% TFA aq. (20 min); flow rate = 5.0 mL/min) to afford 65 (15 mg, 27.3µmol, 66%).
1H NMR (400 MHz, CD2Cl2): δ 0.55 (s, 3H), 0.57 (s, 3H), 2.03 (s, 3H), 2.10 (t, J = 6.9 Hz, 4H), 2.96 (t, J = 7.2 Hz, 2H), 3.24 (s, 3H), 3.57 (t, J = 6.9 Hz, 4H), 3.86 (t, J = 7.2 Hz, 2H), 6.44 (dd, J = 9.2, 2.4 Hz, 1H), 6.78 (s, 1H), 6.93 (s, 1H), 6.98-7.00 (m, 2H), 7.09 (d, J = 7.2 Hz, 1H), 7.33-7.46 (m, 3H); 13C NMR (100 MHz, CD2Cl2) δ –0.99, –0.69, 19.68, 25.77, 26.84, 34.23, 49.30, 55.56, 114.75, 115.43, 121.44, 126.28, 128.10, 129.31, 129.51, 129.61, 130.89, 134.04, 136.40, 139.78, 141.23,
101
147.42, 151.57, 153.47, 158.44, 168.92; HRMS (ESI+) Calcd for [M]+, 437.2413, Found, 437.2387 (–2.6 mmu)
To a solution of 45 (890 mg, 1.57 mmol) in anhydrous THF (15 mL) was added 1 M THF solution of sec-BuLi (3.2 mL, 3.2 mmol) at –78 °C under argon atmosphere. The reaction mixture was stirred for 20 min at the same temperature, then dichlorodimethylsilane (203 µL, 1.70 mmol) in anhydrous THF (5 mL) was added to it and the mixture was warmed to room temperature and stirred for 3 h. 2 N HCl aq. was added to the mixture to quench the reaction, and the mixture was neutralized with NaHCO3 aq. The whole was extracted with CH2Cl2. The organic layer was collected, washed with brine, dried over Na2SO4 and evaporated to dryness. The residue was dissolved in acetone (30 mL) at 0 °C and KMnO4 (187 mg, 1.19 mmol) was added in small portions over 2 h with stirring at the same temperature. Then the mixture was filtered through celite filter and the solution was evaporated to dryness. The residue was purified by column chromatography (silica gel, AcOEt/n-hexane) to afford 56 (77 mg, 0.161 mmol, 10%).
1H NMR (400 MHz, CDCl3): δ 0.45 (s, 6H), 1.49 (s, 9H), 2.91 (s, 3H), 3.06 (t, J = 9.0 Hz, 2H), 3.35 (t, J = 5.0 Hz, 4H), 3.49 (t, J = 9.0 Hz, 2H), 3.62 (t, J = 5.0 Hz, 4H), 6.49 (s, 1H), 7.01-7.04 (m, 2H), 8.20 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ –1.19, 27.88, 28.33, 34.36, 47.56, 54.63, 79.97, 107.74, 116.22, 117.61, 126.02, 130.95, 131.37, 132.20, 132.56, 140.21, 140.29, 151.98, 154.48, 154.91, 184.99; HRMS (ESI+) Calcd for [M+H]+, 478.2526, Found, 478.2483 (–4.3 mmu).
102
To a solution of 2-bromotoluene (36.4 µL, 0.3 mmol) in anhydrous THF (10 mL) was added 1 M cyclohexane solution of sec-butyllithium (0.3 mL, 0.3 mmol) at –78°C. The reaction mixture was maintained for 5 min, and then 56 (50 mg, 105 µmol) in THF (5 mL) was added to it and the mixture was stirred at –78°C for 20 min under argon atmosphere. The reaction mixture was quenched with 2 N HCl aq., diluted with Sat. NaHCO3 aq, and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by HPLC (eluent, 24% CH3CN/0.1% TFA aq. (0 min) to 56% CH3CN/0.1% TFA aq. (20 min); flow rate
= 25.0 mL/min) to afford 66 (13 mg, 28.8 µmol, 27%).
1H NMR (400 MHz, CD3OD): δ 0.58 (s, 3H), 0.60 (s, 3H), 2.03 (s, 3H), 2.95-2.97 (m, 6H), 3.36 (s, 3H), 3.65 (t, J = 5.6 Hz, 4H), 3.95 (t, J = 7.2 Hz, 2H), 6.79 (s, 1H), 6.82 (dd, J = 9.4, 2.8 Hz, 1H), 6.96 (d, J = 9.4 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 7.32 (s, 1H), 7.35-7.47 (m, 4H); 13C NMR (100 MHz, CD3OD) δ –1.53, –1.24, 19.44, 26.71, 34.18, 46.25, 56.39, 115.59, 117.90, 121.58, 126.91, 129.53, 129.94, 130.13, 130.55, 131.39, 134.58, 136.06, 136.96, 140.49, 140.52, 146.29, 154.02, 155.74, 159.95, 167.48; HRMS (ESI+) Calcd for [M]+, 452.2522, Found, 452.2511 (–1.1 mmu); The HPLC chromatogram after purification was as follows. The elution was done with a 20 min linear gradient from 16 % CH3CN/0.1 % TFA aq. to 80 % CH3CN/0.1 % TFA aq. (flow rate = 1.0 mL/min); Absorbance at 640 nm was detected.
To a solution of 6855 (407 mg, 1.08 mmol) in CH2Cl2 (10 ml) were added 1,3-dimethylbarbituric acid (505 mg, 3.24 mmol) and Pd(PPh3)4 (187 mg, 0.162 mmol) under argon atmosphere. The
-1000 19000 39000 59000 79000 99000 119000
0 5 10 15 20
Abs. at 640 nm
Time (min)
103
reaction mixture was stirred for 63 h at 35°C. The reaction was quenched with sat. NaHCO3 aq., and the aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with sat.
NaHCO3 aq. and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, AcOEt/CH2Cl2) to afford 69 (320 mg, 1.08 mmol, quant.).
1H NMR (300 MHz, CDCl3): δ 0.44 (s, 6H), 3.10 (s, 6H), 4.06 (s, 2H), 6.77-6.85 (m, 4H), 8.33 (d, J
= 8.7 Hz, 1H), 8.38 (d, J = 8.7 Hz, 1H); 13C NMR (75 MHz, CDCl3) δ –1.24, 39.96, 113.09, 114.14, 116.11, 117.55, 129.27, 131.69, 131.81, 132.00, 140.34, 140.92, 148.96, 151.46; HRMS (ESI+) Calcd for [M+H]+, 297.1423, Found, 297.1378 (–4.5 mmu)
To a solution of 69 (226 mg, 0.764 mmol) in 5.0 M H2SO4 aq. (12 ml) was added NaNO2 (527 mg, 7.64 mmol) at 0°C. The reaction mixture was stirred for 1 h at the same temperature, and then poured into 2.0 M H2SO4 aq. (50 mL) at 80°C. The reaction mixture was stirred for 10 min, quenched with 2 N NaOH aq. and the aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, AcOEt/CH2Cl2) to afford 70 (95 mg, 0.320 mmol, 42%).
1H NMR (300 MHz, CDCl3): δ 0.46 (s, 6H), 3.11 (s, 6H), 5.22 (s, 3H), 5.30 (s, 3H), 6.78 (d, J = 3.0 Hz, 1H), 6.84 (dd, J = 9.5 Hz, 3.0 Hz, 1H), 6.97 (dd, J = 8.8 Hz, 2.2 Hz, 1H), 7.05 (d, J = 2.2 Hz, 1H), 8.37-8.43 (m, 2H); 13C-NMR (75 MHz, CD3OD) δ –1.3, 40.1, 114.2, 115.6, 118.2, 120.0, 129.6, 132.8, 133.0, 134.1, 142.3, 143.2, 153.5, 161.9, 187.6; LRMS (ESI+) 298 [M+H]+
To a solution of 70 (95 mg, 0.320 mmol) in CH2Cl2 (10 ml) were added
104
N-phenylbis(trifluoromethanesulfonimide) (229 mg, 0.64 mmol) and DIPEA (112 µL, 0.64 mmol) at room temperature. The reaction mixture was stirred for 2 h at the same temperature, quenched with 2 N HCl aq. The aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, AcOEt/CH2Cl2) to afford 71 (120 mg, 0.279 mmol, 87%).
1H NMR (300 MHz, CDCl3): δ 0.51 (s, 6H), 3.13 (s, 6H), 6.77 (d, J = 3.0 Hz, 1H), 6.86 (dd, J = 9.6, 3.0 Hz, 1H), 7.41 (dd, J = 8.7, 2.9 Hz, 1H), 7.48 (d, J = 2.9, 1H), 8.37 (d, J = 9.6, 1H), 8.56 (d, J = 8.7 Hz, 1H)
To a solution of 71 (20.4 mg, 47.5 µmol) in toluene (5 mL) were added bis(trifluoroethyl)amine (172 mg, 0.95 mmol), Pd2(dba)3·CHCl3 (9.8 mg, 9.5 µmol), xantphos (5.5 mg, 9.5 µmol) and Cs2CO3
(310 mg, 0.95 mmol) under argon atmosphere. The reaction mixture was stirred for 39 h at 85°C.
The reaction mixture was diluted with water and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, CH2Cl2/n-hexane) to afford 72 (5 mg, 10.9 µmol, 23%).
1H NMR (300 MHz, CDCl3): δ 0.47 (s, 6H), 3.11 (s, 6H), 4.17 (q, J = 8.0 Hz, 4H), 6.81 (d, J = 2.2 Hz, 1H), 6.86 (dd, J = 9.2, 2.2 Hz, 1), 7.03-7.06 (m, 2H), 8.37-8.44 (m, 2H); HRMS (ESI+) Calcd for [M+H]+, 461.1484, Found, 461.1443 (–4.1 mmu)
105
To a solution of 72 (1.9 mg, 4.13 µmol) in anhydrous THF (4 mL) was added 1 M THF solution of o-tolylmagnesium bromide (0.80 mL, 0.80 mmol), and the mixture was refluxed for 24 h under argon atmosphere. After it had cooled to room temperature, 2 N HCl aq. was added to it. The aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by HPLC (eluent, 56%
CH3CN/0.1% TFA aq. (0 min) to 80% CH3CN/0.1% TFA aq. (20 min); flow rate = 25.0 mL/min) to afford 73 (0.83 mg, 1.25 µmol, 30%).
1H NMR (400 MHz, CD2Cl2): δ 0.59 (s, 6H), 1.95 (s, 6H), 3.40 (s, 3H), 3.54 (s, 3H), 4.25 (q, J = 8.0 Hz, 4H), 6.73 (d, J = 9.6 Hz, 1H), 6.44 (dd, J = 9.6, 3.2 Hz, 1H), 7.14 (d, J = 9.6 Hz, 1H), 7.20 (d, J
= 8.8 Hz, 2H), 7.25-7.28 (m, 3H), 7.35 (t, J = 7.6 Hz, 1H); HRMS (ESI+) Calcd for [M]+, 549.2161, Found, 549.2127 (–3.4 mmu)
To a solution of 66 (12.7 mg, 28.1 µmol) in MeOH (5 mL) were added AcOH (30 µL) and 37%
HCHO aq. (23 µL, 280 µmol). The reaction mixture was stirred for 37 h at room temperature and then added NaCNBH3 (20 mg, 317 µmol). The mixture was stirred for 4.5 h at the same temperature and then added water. The aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue in CH2Cl2 (5 mL) was added p-chloranil (20 mg, 81 µmol). The mixture was stirred for 4 h at room temperature and concentrated in vacuo. The residue purified by HPLC (eluent, 32% CH3CN/0.1% TFA aq. (0 min) to 48% CH3CN/0.1% TFA aq. (20 min); flow rate = 5.0 mL/min) to afford 75 (5.6 mg, 9.66 µmol, 34%).
1H NMR (400 MHz, CD3OD): δ 0.58 (s, 3H), 0.60 (s, 3H), 2.03 (s, 3H), 2.35 (s, 3H), 2.59 (t, J = 5.6 Hz, 4H), 2.98 (t, J = 7.2 Hz, 2H), 3.37 (s, 3H), 3.67 (t, J = 5.6 Hz, 4H), 3.96 (t, J = 7.2 Hz, 2H), 6.80 (s, 1H), 6.83 (dd, J = 9.4, 2.8 Hz, 1H), 6.96 (d, J = 9.4 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 7.34-7.48
106
(m, 5H); 13C NMR (100 MHz, CD3OD) δ –1.55, –1.25, 19.44, 26.70, 34.22, 45.94, 47.18, 55.56, 56.45, 115.39, 118.08, 121.61, 126.92, 129.66, 129.95, 130.13, 130.60, 131.40, 134.65, 136.18, 136.96, 140.40, 140.51, 146.14, 153.88, 155.98, 160.06, 167.49; HRMS (ESI+) Calcd for [M]+, 466.2679, Found, 466.2677 (–0.2 mmu); The HPLC chromatogram after purification was as follows.
The elution was done with a 20 min linear gradient from 24 % CH3CN/0.1 % TFA aq. to 52 % CH3CN/0.1 % TFA aq. (flow rate = 1.0 mL/min); Absorbance at 650 nm was detected.
To a solution of 56 (32 mg, 66.9 µmol) in anhydrous THF (10 mL) was added 1 M THF solution of o-tolylmagnesium bromide (2 mL, 2.0 mmol). The reaction mixture was refluxed for 6 h under argon atmosphere. After it had cooled to room temperature, 2 N HCl aq. was added to it. The aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was added trifluoroacetic acid (4 mL) and stirred for 1 h at room temperature. The mixture was concentrated in vacuo and purified by HPLC (eluent, 29%
CH3CN/0.1% TFA aq. (0 min) to 72% CH3CN/0.1% TFA aq. (20 min); flow rate = 25.0 mL/min) to afford 78 (8.1 mg, 14.0 µmol, 21%).
HRMS (ESI+) Calcd for [M]+, 466.2679, Found, 466.2653 (–2.6 mmu)
107
To a solution of 78 (5 mg, 8.62 µmol) in MeOH (10 mL) were added AcOH (20 µL), 37% HCHO aq.
(1.4 µL, 17.3 µmol) and NaCNBH3 (2.5 mg, 40 µmol) at room temperature. The reaction mixture was stirred for 16 h at the same temperature and then added water. The aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue in CH2Cl2 (5 mL) was added p-chloranil (7 mg, 28.5 µmol). The mixture was stirred for 3 h at room temperature and concentrated in vacuo. The residue purified by HPLC (eluent, 24% CH3CN/0.1% TFA aq. (0 min) to 52% CH3CN/0.1% TFA aq. (20 min); flow rate
= 25.0 mL/min) to afford SiRpH1 (76) (2.0 mg, 2.82 µmol, 37%).
1H NMR (400 MHz, CD3OD): δ 0.59 (s, 6H), 1.98 (s, 6H), 2.35 (s, 3H), 2.59 (t, J = 5.4 Hz, 4H), 2.98 (t, J = 7.2 Hz, 2H), 3.37 (s, 3H), 3.67 (t, J = 5.4 Hz, 4H), 3.97 (t, J = 7.2 Hz, 2H), 6.79 (s, 1H), 6.84 (dd, J = 9.2, 2.6 Hz, 1H), 6.97 (d, J = 9.2 Hz, 1H), 7.22 (d, J = 7.6 Hz, 2H), 7.33-7.37 (m, 2H), 7.39 (d, J = 2.6 Hz, 1H); HRMS (ESI+) Calcd for [M]+, 480.2835, Found, 480.2858 (+2.3 mmu); The HPLC chromatogram after purification was as follows. The elution was done with a 20 min linear gradient from 32 % CH3CN/0.1 % TFA aq. to 64 % CH3CN/0.1 % TFA aq. (flow rate = 1.0 mL/min); Absorbance at 650 nm was detected.
108
To a solution of 78 (3 mg, 5.17 µmol) in CH2Cl2 (5 mL) were added MeOH (1 mL), AcOH (20 µL) and benzaldehyde (1 µL, 9.91 µmol). The reaction mixture was stirred for 10 min at room temperature and then added NaCNBH3 (1.26 mg, 20 µmol). The mixture was stirred for 23 h at the same temperature and then added water. The aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue in CH2Cl2 (10 mL) was added p-chloranil (2 mg, 8.13µmol). The mixture was stirred for 1 h at room temperature and concentrated in vacuo. The residue purified by HPLC (eluent, 24%
CH3CN/0.1% TFA aq. (0 min) to 52% CH3CN/0.1% TFA aq. (20 min); flow rate = 5.0 mL/min) to afford SiRpH2 (79) (1.5 mg, 2.24 µmol, 43%).
1H NMR (400 MHz, CD3OD): δ 0.49 (s, 6H), 1.90 (s, 6H), 2.51 (t, J = 5.0 Hz, 4H), 2.91 (t, J = 7.0 Hz, 2H), 3.25 (s, 3H), 3.49 (s, 2H), 3.54 (t, J = 5.0 Hz, 4H), 3.85 (t, J = 7.0 Hz, 2H), 6.58 (dd, J = 8.6, 2.8 Hz, 1H), 6.72 (s, 1H), 6.89 (d, J = 8.6 Hz, 1H), 6.97 (s, 1H), 7.10-7.12 (m,3H), 7.18-7.28 (m, 6H); HRMS (ESI+) Calcd for [M]+, 556.3148, Found, 556.3134 (–1.4 mmu) ; The HPLC chromatogram after purification was as follows. The elution was done with a 20 min linear gradient from 32 % CH3CN/0.1 % TFA aq. to 64 % CH3CN/0.1 % TFA aq. (flow rate = 1.0 mL/min);
Absorbance at 650 nm was detected.
109
To a solution of 78 (3.5 mg, 6.03 µmol) in MeOH (5 mL) were added AcOH (20 µL), 3-fluorobenzaldehyde (1.3 µL, 12.1 µmol) and NaCNBH3 (1.5 mg, 24.1 µmol) at room temperature.
The reaction mixture was stirred for 16 h at the same temperature and then added water. The aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue in CH2Cl2 (5 mL) was added p-chloranil (3 mg, 12.2µmol). The mixture was stirred for 3 h at room temperature and concentrated in vacuo. The residue was purified by HPLC (eluent, 27% CH3CN/0.1% TFA aq. (0 min) to 64% CH3CN/0.1%
TFA aq. (20 min); flow rate = 25.0 mL/min) to afford SiRpH3 (80) (1.5 mg, 1.87 µmol, 31%).
1H NMR (400 MHz, CD3OD): δ 0.60 (s, 6H), 1.97 (s, 6H), 3.01 (t, J = 6.5 Hz, 2H), 3.38 (brs, 4H), 3.44 (s, 3H), 3.84 (brs, 4H), 4.03 (t, J = 6.5 Hz, 2H), 4.38 (s, 2H), 6.82 (s, 1H), 6.90 (dd, J = 9.4, 3.0 Hz, 1H), 6.99 (d, J = 9.4 Hz, 1H), 7.23-7.38 (m,6H), 7.45-7.56 (m, 3H); HRMS (ESI+) Calcd for [M]+, 574.3054, Found, 574.3032 (–2.2 mmu); The HPLC chromatogram after purification was as follows. The elution was done with a 20 min linear gradient from 32 % CH3CN/0.1 % TFA aq. to 64 % CH3CN/0.1 % TFA aq. (flow rate = 1.0 mL/min); Absorbance at 650 nm was detected.
110
Compound 81 was synthesized from 80 as reported previously56.
To a solution of NaNO2 (1.04 g, 15 mmol) in concentrated H2SO4 aq. (11 ml) were added 4-bromo-2,6-dimethylaniline (80) (3 g, 15 mmol) in AcOH (10 mL) and CuBr (2.58 g, 18 mmol) in 47% HBr aq. (10 mL) at 0°C. The reaction mixture was stirred for 2 h at 70°C and then poured into crashed ice. The aqueous layer was extracted with n-hexane. The combined organic layers were washed with and brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by column chromatography (silica gel, n-hexane) to afford 81 (1.36 g, 5.15 mmol, 34%).
1H NMR (300 MHz, CDCl3): δ2.38 (s, 6H), 7.21 (s, 2H)
To a solution of 81 (1.36 g, 5.15 mmol) in anhydrous THF (15 mL) was added 1.6 M cyclohexane solution of n-butyllithium (3.2 mL, 5.15 mmol) at –78°C. The reaction mixture was maintained for 1.5 h, and then Boc2O (3.37 g, 15.45 mmol) in THF (10 mL) was added to it and the mixture was stirred for 1 h at room temperature under argon atmosphere. The reaction mixture was quenched with 2 N HCl aq., diluted with water and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (silica gel, AcOEt/n-hexane) and GPC to afford 82 (800 mg, 2.81 mmol, 55%).
1H NMR (300 MHz, CDCl3): δ 1.59 (s, 9H), 2.45 (s, 6H), 7.67 (s, 2H); 13C NMR (75 MHz, CDCl3) δ 23.86, 28.14, 81.17, 128.79, 130.19, 132,41, 138.36, 165.48
111
To a solution of 82 (182 mg, 0.638 mmol) in anhydrous THF (10 mL) was added 1 M cyclohexane solution of sec-butyllithium (0.64 mL, 0.64 mmol) at –78°C. The reaction mixture was maintained for 30 min, and then 56 (61 mg, 128 µmol) in THF (5 mL) was added to it and the mixture was stirred at room tempeerature for 3 h under argon atmosphere. The reaction mixture was quenched with 2 N HCl aq., diluted with water and extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was added trifluoroacetic acid (5 mL) and stirred at room temperature for 30 min. The mixture was concentrated in vacuo and purified by HPLC (eluent, 24% CH3CN/0.1% TFA aq. (0 min) to 64%
CH3CN/0.1% TFA aq. (20 min); flow rate = 25.0 mL/min) to afford 83 (50 mg, 80.2 µmol, 63%).
1H NMR (300 MHz, CD3CN): δ 0.56 (s, 6H), 2.00 (s, 6H), 2.91 (t, J = 6.1 Hz, 2H), 3.30 (t, J = 4.5 Hz, 4H), 3.33 (s, 3H), 3.81 (t, J =4.5 Hz, 4H), 3.94 (t, J = 6.1 Hz, 2H), 6.73-6.77 (m, 2H), 6.87 (d, J
= 9.6 Hz, 1H), 7.29 (s, 1H), 7.36 (d, J = 2.4 Hz, 1H), 7.86 (s, 2H); 13C NMR (100 MHz, CD3OD) δ – 1.39, 19.84, 26.39, 35.08, 43.68, 44.41, 56.88, 116.00, 119.30, 121.79, 129.64, 129.99, 132.04, 133.46, 137.28, 137.43, 137.60, 144.22, 144.64, 152.62, 156.24, 160.24, 164.39, 168.28; HRMS (ESI+) Calcd for [M]+, 510.2577, Found, 510.2557 (–2.0 mmu); The HPLC chromatogram after purification was as follows. The elution was done with a 20 min linear gradient from 24 % CH3CN/0.1 % TFA aq. to 56 % CH3CN/0.1 % TFA aq. (flow rate = 1.0 mL/min); Absorbance at 600 nm was detected.
112
To a solution of 83 (16 mg, 25.6 µmol) in MeOH (3 mL) were added AcOH (14.6 µL, 256 µmol), 3-fluorobenzaldehyde (3.3 µL, 30.8 µmol) at room temperature. The reaction mixture was stirred for 10 min at the same temperature and then added NaCNBH3 (1.5 mg, 24.1 µmol). The mixture was stirred for 18 h at room temperature and then added water. The aqueous layer was extracted with CH2Cl2. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. The residue was purified by HPLC (eluent, 27% CH3CN/0.1% TFA aq. (0 min) to 56%
CH3CN/0.1% TFA aq. (20 min); flow rate = 25.0 mL/min) to afford 84 (14.7 mg, 20.1 µmol, 78%).
1H NMR (300 MHz, CD3CN): δ 0.56 (s, 6H), 2.01 (s, 6H), 2.93 (t, J = 6.1 Hz, 2H), 3.20 (brs, 4H), 3.34 (s, 3H), 3.82 (brs, 4H), 3.95 (t, J = 6.1 Hz, 2H), 4.20 (s, 2H), 6.74-6.77 (m, 2H), 6.87 (d, J = 9.6 Hz, 1H), 7.18-7.50 (m, 6H), 7.87 (s, 2H); HRMS (ESI+) Calcd for [M]+, 618.2952, Found, 618.2910 (–4.2 mmu); The HPLC chromatogram after purification was as follows. The elution was done with a 20 min linear gradient from 40 % CH3CN/0.1 % TFA aq. to 64 % CH3CN/0.1 % TFA aq. (flow rate
= 1.0 mL/min); Absorbance at 600 nm was detected.
113
To a solution of 84 (2.3 mg, 3.15 µmol) in DMF (2 mL) were added N-hydroxysuccinimide (NHS) (1.8 mg, 15.8 µmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC•HCl) (3.0 mg, 15.8 µmol) at room temperature. The reaction mixture was stirred for 23 h at the same temperature. The mixture was purified by HPLC (eluent, 36% CH3CN/0.1% TFA aq. (0 min) to 56%
CH3CN/0.1% TFA aq. (20 min); flow rate = 25.0 mL/min) to afford SiRpH3-SE (85) (2.3 mg, 2.77 µmol, 88%).
HRMS (ESI+) Calcd for [M]+, 715.3116, Found, 715.3134 (+1.8 mmu)
To a solution of 83 (18.4 mg, 29.5 µmol) in MeOH (2 mL) were added AcOH (14 µL, 240 µmol), 2-sulfobenzaldehyde sodium salt (10 mg, 48 µmol) and NaCNBH3 (2.2 mg, 35.4 µmol) at room temperature. The reaction mixture was stirred for 19 h at the same temperature. The mixture was purified by HPLC (eluent, 24% CH3CN/0.1% TFA aq. (0 min) to 56% CH3CN/0.1% TFA aq. (20 min); flow rate = 5.0 mL/min) to afford SiRpH4 (86) (4.4 mg, 5.55 µmol, 19%).
1H NMR (300 MHz, CD3OD): δ 0.61 (s, 6H), 2.05 (s, 6H), 3.02 (t, J = 6.6 Hz, 2H), 3.46 (s, 3H), 3.54 (brs, 4H), 4.05 (t, J = 6.6 Hz, 2H), 4.36 (brs, 4H), 4.68 (s, 2H), 6.76 (s, 2H), 6.92-6.93 (m, 2H), 7.48-7.63 (m, 5H), 7.90 (s, 2H), 8.06 (dd, J = 6.6, 2.1 Hz, 1H); HRMS (ESI+) Calcd for [M]+, 680.2614, Found, 680.2580 (–3.4 mmu) ; The HPLC chromatogram after purification was as follows.
The elution was done with a 20 min linear gradient from 24 % CH3CN/0.1 % TFA aq. to 56 % CH3CN/0.1 % TFA aq. (flow rate = 1.0 mL/min); Absorbance at 600 nm was detected.
114
To a solution of 83 (12.8 mg, 20.5 µmol) in MeOH (5 mL) were added AcOH (250 µL) and disodium 4-formylbenzene-1,3-disulfonate (19.1 mg, 61.5 µmol) at room temperature. The reaction mixture was stirred for 30 min at the same temperature and then added 2-picoline borane (4.4 mg, 41 µmol). The mixture was stirred for 16 h at room temperature and purified by HPLC (eluent, 27%
CH3CN/0.1% TFA aq. (0 min) to 60% CH3CN/0.1% TFA aq. (20 min); flow rate = 25.0 mL/min) to afford SiRpH5 (87) (11.2 mg, 12.8 µmol, 63%).
1H NMR (400 MHz, 20 mM pD 9.7 phosphate buffered D2O): δ 0.48 (s, 6H), 1.87 (s, 6H), 2.63 (brs, 4H), 2.81 (t, J = 7.0 Hz, 2H), 3.31 (s, 3H), 3.50 (brs, 4H), 3.90 (t, J = 7.0 Hz, 2H), 4.00 (s, 2H), 6.51 (d, J = 9.4 Hz, 1H), 6.75 (s, 1H), 6.84 (d, J = 9.4 Hz, 1H), 7.28 (s, 1H), 7.38 (s, 1H), 7.65 (s, 2H), 7.72 (d, J = 8.2 Hz, 1H), 7.88 (d, J = 8.2 Hz, 1H), 8.30 (s, 1H); 13C NMR (100 MHz, CD3OD:D2O = 1:3) δ –1.11, 20.10, 26.73, 35.45, 45.53, 52.91, 57.57, 59.77, 117.07, 119.58, 120.19, 122.40, 126.87, 129.41, 130.21, 130.34, 130.81, 131.79, 133.57, 135.96, 137.56, 138.12, 138.47, 144.92, 145.32, 146.11, 147.92, 152.72, 157.61, 160.90, 163.91, 170.85; HRMS (ESI+) Calcd for [M]+, 760.2183, Found, 760.2137 (–4.6 mmu) ; The HPLC chromatogram after purification was as follows. The elution was done with a 20 min linear gradient from 24 % CH3CN/0.1 % TFA aq. to 56 % CH3CN/0.1 % TFA aq. (flow rate = 1.0 mL/min); Absorbance at 600 nm was detected.
-3000 97000 197000 297000 397000 497000 597000
0 5 10 15 20
Abs. at 600 nm
Time (min)
115
To a solution of SiRpH5 (87) (11.2 mg, 12.8 µmol) in DMF (2 mL) were added N-hydroxysuccinimide (NHS) (2.2 mg, 18.9 µmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC•HCl) (3.6 mg, 18.9 µmol) and DIPEA (6.6 µL, 37.8 µmol) at room temperature.
The reaction mixture was stirred for 20 h at the same temperature, purified by HPLC (eluent, 27%
CH3CN/0.1% TFA aq. (0 min) to 60% CH3CN/0.1% TFA aq. (20 min); flow rate = 25.0 mL/min) and concentrated in vacuo. The residue was added DMF (2 mL), NH2-PEG6-OH (4.9 mg, 13.7 µmol), DIPEA (25 µL, 260 µmol) and H2O (50 µL) at room temperature. The he reaction mixture was stirred for 15 h at the same temperature, purified by HPLC (eluent, 27% CH3CN/0.1% TFA aq. (0 min) to 60% CH3CN/0.1% TFA aq. (20 min); flow rate = 25.0 mL/min) and concentrated in vacuo to afford 88 as a crude material. The residue was added DMF (3 mL), NHS (4 mg, 35 µmol), EDC•HCl (6.7 mg, 35 µmol) and DIPEA (20 µL, 208 µmol) at room temperature. The reaction mixture was stirred for 17 h at the same temperature, purified by HPLC (eluent, 24% CH3CN/0.1% TFA aq. (0 min) to 44% CH3CN/0.1% TFA aq. (20 min); flow rate = 25.0 mL/min) to afford SiRpH5-PEG6-SE
116 (89) (4.1 mg, 3.44 µmol, 27% in 3 steps).
HRMS (ESI+) Calcd for [M]+, 1192.4290, Found, 1192.4270 (–2.0 mmu)
Cartesian Coordinates and Total Electron Energies
Non-protonated form of SiR (75)
LUMO –3.30 eV HOMO –5.65 eV
E(RB3LYP) = -1618.47647643 a.u.
--- Center Atomic Atomic Coordinates (Angstroms) Number Number Type X Y Z
--- 1 6 0 -2.179988 -0.940013 0.173451 2 6 0 -1.299449 1.675608 -0.002391 3 6 0 -3.160939 0.103976 0.167054 4 6 0 -2.649056 1.433711 0.086159 5 1 0 -2.514784 -1.965908 0.271733 6 1 0 -0.978919 2.707696 -0.080550 7 6 0 -0.818406 -0.706867 0.095122 8 6 0 1.390675 2.448173 -0.205529 9 6 0 2.188257 0.082255 -0.079897 10 6 0 -0.317545 0.636728 -0.002096 11 6 0 2.057869 -1.350678 0.022624 12 6 0 1.073163 0.981062 -0.086140 13 6 0 4.475224 -1.589466 -0.058071 14 6 0 1.459610 3.018406 -1.485573 15 6 0 1.615577 3.244605 0.939007 16 6 0 4.621455 -0.178156 -0.161057 17 6 0 3.519787 0.628955 -0.173002 18 6 0 3.184588 -2.162019 0.032164 19 6 0 1.975453 5.170232 -0.524905 20 6 0 1.905893 4.604333 0.750361 21 6 0 1.751430 4.374165 -1.650022 22 1 0 3.080693 -3.242010 0.095993 23 1 0 3.648306 1.702173 -0.255850 24 1 0 2.080185 5.228666 1.623575 25 1 0 2.202702 6.226823 -0.636949 26 1 0 1.800964 4.801064 -2.647862 27 1 0 -3.315210 2.285896 0.065856 28 7 0 5.680059 -2.200656 -0.063779
117
29 14 0 0.363291 -2.170203 0.119752 30 6 0 0.088037 -3.277509 -1.386120 31 1 0 0.816053 -4.097973 -1.410197 32 1 0 -0.912213 -3.728079 -1.367999 33 1 0 0.185370 -2.714128 -2.321152 34 6 0 0.192145 -3.157294 1.720928 35 1 0 -0.809533 -3.596424 1.808547 36 1 0 0.913982 -3.982880 1.754588 37 1 0 0.360390 -2.527559 2.601933 38 6 0 6.100726 0.150051 -0.211464 39 1 0 6.422987 0.638212 0.716394 40 1 0 6.360717 0.818143 -1.037937 41 6 0 6.764464 -1.243238 -0.363028 42 1 0 7.599742 -1.401309 0.326464 43 1 0 7.130845 -1.417349 -1.384808 44 6 0 5.916180 -3.631188 -0.116393 45 1 0 5.145750 -4.163684 0.446350 46 1 0 5.924291 -4.001789 -1.151751 47 1 0 6.885351 -3.852091 0.340036 48 1 0 1.282481 2.392767 -2.357336 49 6 0 1.551681 2.665116 2.335024 50 1 0 0.574199 2.212844 2.543632 51 1 0 2.304850 1.880920 2.483489 52 1 0 1.726841 3.440616 3.086561 53 6 0 -5.044583 -1.522930 0.267747 54 6 0 -5.497156 0.856502 0.604919 55 6 0 -6.361587 -1.619688 -0.507929 56 1 0 -5.208230 -1.816006 1.315361 57 1 0 -4.339165 -2.219425 -0.182961 58 6 0 -6.794807 0.697559 -0.186542 59 1 0 -5.710084 0.741094 1.678007 60 1 0 -5.103578 1.857392 0.444976 61 1 0 -6.765260 -2.628825 -0.368492 62 1 0 -6.155065 -1.488832 -1.590321 63 1 0 -7.521944 1.422048 0.196788 64 1 0 -6.609264 0.940441 -1.253079 65 7 0 -4.498584 -0.157677 0.222948 66 7 0 -7.330164 -0.647167 -0.024158 67 6 0 -8.638637 -0.805865 -0.652725 68 1 0 -9.018659 -1.813645 -0.457009 69 1 0 -9.341448 -0.086883 -0.219865 70 1 0 -8.608966 -0.649804 -1.747829 ---
118 Protonated form of SiR (75)
LUMO –3.48 eV HOMO –6.04 eV
E(RB3LYP) = -1618.78140098 a.u.
--- Center Atomic Atomic Coordinates (Angstroms) Number Number Type X Y Z
--- 1 6 0 -2.173450 -0.904784 -0.013054 2 6 0 -1.228423 1.691143 -0.115033 3 6 0 -3.103367 0.155889 -0.042469 4 6 0 -2.599139 1.464997 -0.091735 5 1 0 -2.549456 -1.922704 0.066652 6 1 0 -0.885526 2.717047 -0.169783 7 6 0 -0.794698 -0.696973 -0.034368 8 6 0 1.466316 2.442515 -0.218384 9 6 0 2.227399 0.076238 -0.087941 10 6 0 -0.284492 0.636463 -0.089824 11 6 0 2.075554 -1.369706 -0.006493 12 6 0 1.145125 0.976323 -0.120727 13 6 0 4.494912 -1.626181 -0.002867 14 6 0 1.584744 3.017809 -1.493516 15 6 0 1.644236 3.232281 0.939378 16 6 0 4.664770 -0.206455 -0.088193 17 6 0 3.578793 0.608981 -0.129221 18 6 0 3.185002 -2.187355 0.036067 19 6 0 2.057964 5.165196 -0.500971 20 6 0 1.939394 4.593462 0.767996 21 6 0 1.880727 4.374710 -1.638613 22 1 0 3.069721 -3.265775 0.097470 23 1 0 3.718039 1.681729 -0.195933 24 1 0 2.081327 5.213622 1.649736 25 1 0 2.289632 6.221993 -0.598807 26 1 0 1.972785 4.806413 -2.631143 27 1 0 -3.260259 2.323418 -0.136179 28 7 0 5.677306 -2.246453 0.026448 29 14 0 0.367971 -2.176732 0.034713 30 6 0 0.137393 -3.281941 -1.477665 31 1 0 0.860995 -4.106431 -1.475786 32 1 0 -0.863200 -3.732089 -1.490822 33 1 0 0.268519 -2.723700 -2.411529 34 6 0 0.133989 -3.144285 1.637332
119
35 1 0 -0.871300 -3.580419 1.692807 36 1 0 0.849223 -3.973279 1.705725 37 1 0 0.272980 -2.508236 2.518785 38 6 0 6.147078 0.104033 -0.123200 39 1 0 6.447493 0.750617 0.707594 40 1 0 6.429450 0.615211 -1.049512 41 6 0 6.810804 -1.293652 -0.023805 42 1 0 7.418596 -1.416443 0.879856 43 1 0 7.439990 -1.533515 -0.887982 44 6 0 5.937816 -3.674874 0.118042 45 1 0 5.004370 -4.237592 0.108886 46 1 0 6.552452 -3.993858 -0.731111 47 1 0 6.479242 -3.897440 1.044884 48 1 0 1.446686 2.397012 -2.375918 49 6 0 1.538471 2.643791 2.329374 50 1 0 0.577155 2.139808 2.490112 51 1 0 2.324738 1.901092 2.516856 52 1 0 1.635388 3.423379 3.090301 53 6 0 -5.407230 0.993379 0.255721 54 6 0 -5.045187 -1.066559 -0.947962 55 6 0 -6.748788 0.483735 0.778843 56 1 0 -5.565606 1.606841 -0.651546 57 1 0 -4.997131 1.651030 1.026208 58 6 0 -6.374506 -1.636653 -0.460715 59 1 0 -5.181374 -0.588932 -1.937139 60 1 0 -4.360592 -1.903002 -1.094927 61 1 0 -7.463658 1.302204 0.897395 62 1 0 -6.631061 -0.037070 1.732494 63 1 0 -6.826041 -2.291780 -1.210461 64 1 0 -6.256655 -2.183644 0.478325 65 7 0 -4.494621 -0.118295 0.020533 66 6 0 -8.693835 -1.028365 0.290320 67 1 0 -9.364988 -0.179832 0.435180 68 1 0 -9.099706 -1.705044 -0.463947 69 1 0 -8.548487 -1.558592 1.232898 70 1 0 -7.518375 -0.025765 -1.071931 71 7 0 -7.358567 -0.516824 -0.182923
---120
参考文献
1. J. K. Willmann, N. van Bruggen, L. M. Dinkelborg and S. S. Gambhir, Nat. Rev.
Drug Discov., 2008, 7, 591-607.
2. Y. Urano, M. Sakabe, N. Kosaka, M. Ogawa, M. Mitsunaga, D. Asanuma, M. Kamiya, M. R. Young, T. Nagano, P. L. Choyke and H. Kobayashi, Sci. Transl. Med., 2011, 3, 110ra119.
3. R. Weissleder, Nat Biotech, 2001, 19, 316-317.
4. J. V. Frangioni, Curr. Opin. Chem. Biol., 2003, 7, 626-634.
5. K. L. Arendt, M. Royo, M. Fernandez-Monreal, S. Knafo, C. N. Petrok, J. R. Martens and J. A. Esteban, Nat. Neurosci., 2010, 13, 36-44.
6. T. Egawa, K. Hanaoka, Y. Koide, S. Ujita, N. Takahashi, Y. Ikegaya, N. Matsuki, T.
Terai, T. Ueno, T. Komatsu and T. Nagano, J. Am. Chem. Soc., 2011, 133, 14157-14159.
7. M. Mizunuma, H. Norimoto, K. Tao, T. Egawa, K. Hanaoka, T. Sakaguchi, H. Hioki, T. Kaneko, S. Yamaguchi, T. Nagano, N. Matsuki and Y. Ikegaya, Nat. Neurosci., 2014, 17, 503-505.
8. Y. Koide, Y. Urano, K. Hanaoka, T. Terai and T. Nagano, J. Am. Chem. Soc., 2011, 133, 5680-5682.
9. M. Fu, Y. Xiao, X. Qian, D. Zhao and Y. Xu, Chem. Commun., 2008, DOI:
10.1039/b718544h, 1780-1782.
10. Y. Kushida, T. Nagano and K. Hanaoka, Analyst, 2015, 140, 685-695.
11. Y. Koide, Y. Urano, K. Hanaoka, T. Terai and T. Nagano, ACS Chem. Biol., 2011, 6, 600-608.
12. E. D’Este, D. Kamin, F. Göttfert, A. El-Hady and Stefan W. Hell, Cell Reports, 2015, 10, 1246-1251.
13. H. Sasaki, K. Hanaoka, Y. Urano, T. Terai and T. Nagano, Bioorg. Med. Chem., 2011, 19, 1072-1078.
14. J. Lee, K. H. Lee, J. Jeon, A. Dragulescu-Andrasi, F. Xiao and J. Rao, ACS Chem.
Biol., 2010, 5, 1065-1074.
15. C. N. Im, N. Y. Kang, H. H. Ha, X. Bi, J. J. Lee, S. J. Park, S. Y. Lee, M. Vendrell, Y.
K. Kim, J. S. Lee, J. Li, Y. H. Ahn, B. Feng, H. H. Ng, S. W. Yun and Y. T. Chang, Angew. Chem. Int. Ed., 2010, 49, 7497-7500.
16. Y. Koide, Y. Urano, K. Hanaoka, W. Piao, M. Kusakabe, N. Saito, T. Terai, T. Okabe and T. Nagano, J. Am. Chem. Soc., 2012, 134, 5029-5031.
121
17. B. Wang, X. Chai, W. Zhu, T. Wang and Q. Wu, Chem. Commun., 2014, 50, 14374-14377.
18. Y. Kushida, K. Hanaoka, T. Komatsu, T. Terai, T. Ueno, K. Yoshida, M. Uchiyama and T. Nagano, Bioorg. Med. Chem. Lett., 2012, 22, 3908-3911.
19. J. B. Grimm, B. P. English, J. Chen, J. P. Slaughter, Z. Zhang, A. Revyakin, R. Patel, J. J. Macklin, D. Normanno, R. H. Singer, T. Lionnet and L. D. Lavis, Nat Meth, 2015, 12, 244-250.
20. W. Piao, S. Tsuda, Y. Tanaka, S. Maeda, F. Liu, S. Takahashi, Y. Kushida, T.
Komatsu, T. Ueno, T. Terai, T. Nakazawa, M. Uchiyama, K. Morokuma, T. Nagano and K. Hanaoka, Angew. Chem. Int. Ed., 2013, 52, 13028-13032.
21. T. Peng, N. K. Wong, X. Chen, Y. K. Chan, D. H. Ho, Z. Sun, J. J. Hu, J. Shen, H.
El-Nezami and D. Yang, J. Am. Chem. Soc., 2014, 136, 11728-11734.
22. A. Kumar, M. Kumar and M. K. Gupta, Tetrahedron Lett., 2009, 50, 7024-7027.
23. H. Takahashi, N. Kashiwa, H. Kobayashi, Y. Hashimoto and K. Nagasawa, Tetrahedron Lett., 2002, 43, 5751-5753.
24. Y.-Y. Lai, N.-T. Lin, Y.-H. Liu, Y. Wang and T.-Y. Luh, Tetrahedron, 2007, 63, 6051-6055.
25. C.-Y. Tsai, R. Sung, B.-R. Zhuang and K. Sung, Tetrahedron, 2010, 66, 6869-6872.
26. J. B. Grimm and L. D. Lavis, Org. Lett., 2011, 13, 6354-6357.
27. P. Autissier, C. Soulas, T. H. Burdo and K. C. Williams, Cytometry. Part A : the journal of the International Society for Analytical Cytology, 2010, 77, 410-419.
28. S. P. Perfetto, P. K. Chattopadhyay and M. Roederer, Nat. Rev. Immunol., 2004, 4, 648-655.
29. H. Kobayashi, M. Ogawa, R. Alford, P. L. Choyke and Y. Urano, Chem. Rev., 2010, 110, 2620-2640.
30. 朴文, 博士論文, 東京大学・薬学系研究科, 2015.
31. 明珍琢也, 博士論文, 東京大学・薬学系研究科, 2014.
32. C. E. Lin, Y. Deng, Jr., W. S. Liao, S. W. Sun, W. Y. Lin and C. C. Chen, J. Chromatogr.
A, 2004, 1051, 283-290.
33. D. Asanuma, Y. Takaoka, S. Namiki, K. Takikawa, M. Kamiya, T. Nagano, Y. Urano and K. Hirose, Angew. Chem. Int. Ed., 2014, 53, 6085-6089.
34. A. P. de Silva, T. S. Moody and G. D. Wright, Analyst, 2009, 134, 2385-2393.
35. J. R. Casey, S. Grinstein and J. Orlowski, Nat. Rev. Mol. Cell Biol., 2010, 11, 50-61.
36. J. Han and K. Burgess, Chem. Rev., 2010, 110, 2709-2728.
37. K. Teter, G. Chandy, B. Quinones, K. Pereyra, T. Machen and H. P. Moore, J. Biol.
Chem., 1998, 273, 19625-19633.
122
38. D. J. Yamashiro, B. Tycko, S. R. Fluss and F. R. Maxfield, Cell, 1984, 37, 789-800.
39. T. Myochin, K. Kiyose, K. Hanaoka, H. Kojima, T. Terai and T. Nagano, J. Am. Chem.
Soc., 2011, 133, 3401-3409.
40. J. Van Eerden, M. Skowronska-Ptasinska, P. D. J. Grootenhuis, S. Harkema and D.
N. Reinhoudt, J. Am. Chem. Soc., 1989, 111, 700-706.
41. M. L. Circu and T. Y. Aw, Biochim. Biophys. Acta, 2012, 1823, 1767-1777.
42. P. M. Haggie and A. S. Verkman, J. Biol. Chem., 2009, 284, 7681-7686.
43. B. Poole and S. Ohkuma, J. Cell Biol., 1981, 90, 665-669.
44. I. De Domenico, D. McVey Ward and J. Kaplan, Nat. Rev. Mol. Cell Biol., 2008, 9, 72-81.
45. D. J. Yamashiro and F. R. Maxfield, J. Cell Biol., 1987, 105, 2723-2733.
46. R. Gagescu, N. Demaurex, R. G. Parton, W. Hunziker, L. A. Huber and J. Gruenberg, Mol. Biol. Cell, 2000, 11, 2775-2791.
47. R. Misaki, T. Nakagawa, M. Fukuda, N. Taniguchi and T. Taguchi, Biochem. Biophys.
Res. Commun., 2007, 360, 580-585.
48. S. H. Fan, Y. Numata and M. Numata, Mol. Biol. Cell, 2015, DOI:
10.1091/mbc.E15-04-0257.
49. E. Itakura, C. Kishi-Itakura and N. Mizushima, Cell, 2012, 151, 1256-1269.
50. A. D. Becke, J. Chem. Phys., 1993, 98, 1372-1377.
51. A. D. Becke, J. Chem. Phys., 1993, 98, 5648-5652.
52. C. Lee, W. Yang and R. G. Parr, Physical review. B, Condensed matter, 1988, 37, 785-789.
53. Gaussian 09, Revision A.02, M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E.
Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G. A.
Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian, A. F. Izmaylov, J.
Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J.
Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J. A.
Montgomery Jr., J. E. Peralta, F. Ogliaro, M. J. Bearpark, J. Heyd, E. N. Brothers, K.
N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. P.
Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, N. Rega, N. J. Millam, M.
Klene, J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E.
Stratmann, O. Yazyev, A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L.
Martin, K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S.
Dapprich, A. D. Daniels, Ö. Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski and D.
J. Fox, Gaussian, Inc., Wallingford, CT, USA, 2009.
54. T. Egawa, Y. Koide, K. Hanaoka, T. Komatsu, T. Terai and T. Nagano, Chem.
123 Commun., 2011, 47, 4162-4164.
55. T. Nagano, K. Hanaoka, T. Egawa, Y. Kushida, K. Numasawa, T. Myochin and W.
Piao, WO 2014/106957 A1, 2014.
56. B. Kang, J. W. Kurutz, K.-T. Youm, R. K. Totten, J. T. Hupp and S. T. Nguyen, Chemical Science, 2012, 3, 1938-1944.